F ACULTAD DE C IENCIAS – D EPARTAMENTO DE B IOLOGÍA M OLECULAR
“T HE QUASI - ATOMIC STRUCTURE OF HUMAN TYROSINE HYDROXYLASE BY CRYO - ELECTRON
MICROSCOPY : FUNCTIONAL IMPLICATIONS ”
M ARÍA T ERESA B UENO C ARRASCO
M ADRID , 2019
F ACULTAD DE C IENCIAS – D EPARTAMENTO DE B IOLOGÍA M OLECULAR
Memoria presentada para optar al grado de Doctora por:
M ARÍA T ERESA B UENO C ARRASCO Licenciada en Bioquímica
M ADRID , 2019
DIRECTORES: JOSÉ MARÍA VALPUESTA MORALEJO
CENTRO NACIONAL DE BIOTECNOLOGÍA
JORGE CUÉLLAR PÉREZ
CENTRO NACIONAL DE BIOTECNOLOGÍA
The work described in this thesis was carried out in the
“Structure and Function of Molecular Chaperones”
laboratory of the Macromolecular Structure department at the National Centre for Biotechnology-CSIC in Madrid; and was financially supported by an FPI grant from the Spanish Economy Ministry.
Me gustaría agradecer en primer lugar a mis directores de tesis, José María Valpuesta y Jorge Cuéllar, por darme la oportunidad de trabajar en su laboratorio y por codirigir este trabajo y enseñarme tanto a lo largo de estos años. A Elías Herrero por dirigirme en mis primeros pasos en el laboratorio junto con Virginia Rodríguez, y a Charo Fernández por toda su ayuda y su disposición en cualquier momento.
Quisiera agradecer a Aurora Martínez y a Marte Flydal por facilitarnos las proteínas y todo el intercambio de correos e ideas que han hecho tan fácil y amena la colaboración y la realización de este trabajo.
Todo el trabajo desempeñado no hubiera sido posible sin la ayuda de todas las personas involucradas en los servicios. Gracias a Rocío Arranz, Javier Chichón y Rafael Núñez, integrantes del servicio de criomicroscopía del CNB, así como a Cristina Patiño, Beatriz Martín y Maite Rejas de los servicios de microscopía del CNB y CBM.
A César Santiago, Luis Alberto Campos, Rafael Fernández-Leiro, Miguel Marcilla, José Ramón López-Blanco y Pablo Chacón por la colaboración y ayuda en todos los últimos pasos de este trabajo.
A todo el equipo de Scipion por estar siempre dispuestos a resolver dudas y sobre todo, problemas.
A Barry Willardson por darme la oportunidad de trabajar en su laboratorio. Gracias Grant Ludlam por enseñarme y acompañarme en mis días allí.
A José L. Carrascosa, Jaime Martín-Benito, José Jesús Fernández, Carmen Sanmartín y José R.
Castón por tener siempre abiertas las puertas de sus laboratorios.
Por último, al resto de servicios del CNB, que me han ayudado durante estos años:
instrumentación, divulgación, viajes, compras, personal, servicios generales y almacén.
The aromatic amino acid hydroxylases (AAAHs) constitute a family of enzymes that catalyse the hydroxylation of aromatic amino acids using tetrahydrobiopterin (BH4) as cofactor and di-oxygen as additional substrate. Tyrosine hydroxylase (TH) is an AAAH that catalyses the conversion of L-tyrosine to L-DOPA, the first and rate-limiting step in the biosynthesis of catecholamine neurotransmitters (dopamine, noradrenaline and adrenaline). TH is a highly controlled enzyme, and the regulatory mechanisms include feed-back inhibition by catecholamine end products and phosphorylation at four different Ser/Thr sites. Mutations in TH are associated with a neuropsychiatric disorder characterized by a large reduction in dopamine and noradrenaline levels, and a metabolic phenotype that is also observed in the non-motor and motor symptoms of the neurodegenerative disease Parkinson’s disease (PD).
TH is a 224 kDa homotetramer built by two dimers (D2 symmetry). Each subunit consists of a regulatory ACT domain with an unstructured N-terminal tail, a catalytic domain and a C-terminal oligomerization domain. To date, only structures of truncated forms of the protein are available, such as the crystal structure of the catalytic and oligomerization domains or the solution structure of regulatory domain.
Improvements in the purification process and sample preparation have allowed to obtain an active, full-length TH with an intact N-terminus.
In this work, we have used this kind of preparation to generate the structure of the full-length human TH at 3.4 Å resolution, by means of state-of-the-art cryo- electron microscopy. This structure confirms that the regulatory domain is in a dimeric form but shows slight differences with regard to the previous, partial structures of TH.
A similar approach has been used to determine the structure of TH with dopamine bound to the active site, which in this case has served to visualise for the first time an a-helix of the unstructured, flexible N-terminal part, but which is found internalized in the active site. These structural changes serve to explain, in structural terms, the biochemical results obtained previously, and to have a more comprehensive understanding of the hydroxylation mechanism of TH and its regulatory properties.
Las hidroxilasas de aminoácidos aromáticos (AAAHs) constituyen una familia de enzimas que catalizan la hidroxilación de aminoácidos aromáticos usando tetrahidrobiopterina (BH4) como cofactor y oxígeno como sustrato adicional. La tirosina hidroxilasa (TH) es una AAAH que cataliza la conversión de L-tirosina a L-DOPA, la primera reacción que constituye el paso limitante en la biosíntesis de catecolaminas (dopamina, noradrenalina y adrenalina). TH es una enzima altamente controlada, y los mecanismos de regulación incluyen inhibición por retroalimentación negativa mediante la unión de catecolaminas, y fosforilaciones en cuatro residuos Ser/Thr específicos. Mutaciones en TH se asocian a patologías neurológicas caracterizadas por una reducción en los niveles de dopamina y noradrenalina, y un fenotipo metabólico característico en síntomas motores y no motores observados en la enfermedad de Parkinson.
TH es un homotetrámero de 224 kDa formado por dos dímeros con simetría D2.
Cada subunidad está formada por un dominio ACT regulatorio con una región N- terminal desordenada, un dominio catalítico y un dominio de oligomerización en el extremo C-terminal. Hasta la fecha, solo versiones truncadas de la proteína han sido resueltas, como la estructura atómica de los dominios catalíticos y de oligomerización, o la de los dominios regulatorios. Mejoras en el proceso de purificación y en la preparación de las muestras de TH han permitido obtener una proteína activa y con un extremo N-terminal intacto.
En este trabajo, hemos obtenido la estructura de TH humana completa a una resolución final de 3.4 Å mediante crio-microscopía electrónica. Esta estructura confirma que el dominio regulatorio dimeriza pero muestra ligeras diferencias con respecto a estructuras parciales previas de TH.
Procedimientos similares se han utlizado para determinar la estructura de TH con dopamina unida a su centro activo, en la cual se ha visto por primera vez la a-hélice del N-terminal desestructurado, la cual se internaliza en el sitio activo. Estos cambios estructurales explican los resultados bioquímicos obtenidos en trabajos previos, y ayudan a tener un mayor conocimiento sobre el mecanismo de hidroxilación de TH y sus propiedades regulatorias.
1. Introduction 7
1.1. Aromatic Amino Acid Hydroxylases 9
1.1.1. Evolution of the AAAHs 11
1.1.2. Function of the AAAHs 11
1.1.3. Structure of the AAAHs 12
1.1.4. AAAHs substrate specificity 14
1.1.5. Regulation of AAAHs activity 15
1.1.6. Catalytic reaction 16
1.2. Tyrosine hydroxylase 18
1.2.1. Structure of TH 19
1.2.2. Function and regulation 22
1.2.3. Dysfunction and associated diseases 26
1.3. The RD, a key factor in TH modulation 28
2. Objectives 31
3. Materials and methods 35
3.1. Biological material and reagents 37
3.2. Dopamine binding 37
3.3. Sample preparation for Electron Microscopy 38
3.3.1. Negative staining grids preparation 38
3.3.2. Cryo-EM grids preparation 38
3.4. Cryo-EM 38
3.4.1. Data acquisition 38
3.4.2. Image processing, particle selection and three- dimensional analysis
40
3.5. Model Building, refinement and validation 42
3.6. Atomic structure and loop modelling 43
3.7. Circular dichroism spectroscopy (CDS) 46
3.8. Crosslinking experiments and Mass Spectrometry analysis 46
4. Results 49 4.1. Structural characterization of human apo-TH 51 4.1.1. 3D reconstruction and model building 53 4.2. Structural characterization of human TH(DA) 64 4.2.1. 3D reconstruction and model building 65 4.3. Structural comparison between apo-TH and TH(DA) 72
5. Discussion 79
5.1. Structural arrangement of human apo-TH 81
5.2. New features in the CD of the atomic structure of human apo-TH 83 5.3. Structural rearrangements of TH bound to DA (TH(DA)) 85
5.4. Cryo-EM insights in AAAHs 87
5.5. Structural and functional implications 89
6. Conclusions 91
7. Bibliography 97
Table 1 TH genetic dysfunctions and associated diseases Table 2 Data collection parameters of apo-TH and TH(DA)
Table 3 Apo-TH model refinement and statistics of the CD and OD Table 4 TH(DA) model refinement and statistics of the CD and OD Table 5 XL-MS analysis of apo-TH and TH(DA)
F IGURE I NDEX
Figure 1 The function of aromatic amino acid hydroxylases (AAAHs) Figure 2 Overall domain structure of the AAAHs
Figure 3 Schematic reaction mechanism of the AAAHs Figure 4 Human TH isoforms
Figure 5 Structural organization of TH
Figure 6 Regulation of TH by phosphorylation
Figure 7 Overall scheme of 3D reconstruction maps by cryo-EM Figure 8 Sample preparation and analysis for cryo-EM
Figure 9 First steps in image processing
Figure 10 “De novo” initial models generated with EMAN Figure 11 First steps in 3D reconstruction of apo-TH Figure 12 Symmetry imposition in apo-TH
Figure 13 Final steps in 3D reconstruction of apo-TH
Figure 14 Resolution improvement by subtraction and masking Figure 15 Model sharpening
Figure 16 Density map of apo-TH showing the active site Figure 17 First steps in image processing of TH(DA)
Figure 18 3D reconstruction of TH(DA) containing both RD and CD Figure 19 CD cryo-EM map of TH(DA)
Figure 20 Atomic model prediction of TH(DA)
Figure 21 Structural comparison of apo-TH and TH(DA)
Figure 22 CDS comparative spectra profiles of apo-TH, TH(DA), THDN43(DA) and THDN70(DA)
Figure 23 Model of the proposed rearrangement of the N-terminal region of TH upon DA binding based on the XL-MS experiment
Figure 24 RD arrangement
Figure 25 CD comparison of the active site of different TH atomic structures Figure 26 Comparison between TH and PAH
2D Two-dimensional
3D Three-dimensional
4a-OH-BH4 4a-hydroxypterin
Å Angstrom
AAAHs Aromatic Amino Acid Hydroxylases
ACT domain Aspartate kinase-Chorismate mutase-TyrA domain
AD Adrenaline
AD-HPD/DRD Autosomal dominant hereditary progressive and Dopa-responsive dystonia
AR-DRD Autosomal Recessive Dopa-Responsive Dystonia AR-JP Autosomal recessive juvenile parkinsonism BH4 (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin BS3 Bis(sulfosuccinimidyl suberate)
C-terminal Carboxyl terminal
CA Catecholamine
CaMPKII Ca+/calmodulin-dependent protein kinase II
CD Catalytic domain
Cdk5 Cell division protein kinase 5
CDS Circular Dichroism Spectroscopy
CNS Central nervous system
Cryo-EM Cryo-electron microscopy
CTF Contrast transfer function
DA Dopamine
DRD Dopa responsive dystonia
EM Electron microscopy
ERK Extracellular-signal-regulated kinase
FAS Ferrous ammonium sulphate
FDR False discovery rate
Fe (II) Ferrous iron
Fe (III) Ferric iron
FSC Fourier shell correlation
HPLC High pressure liquid chromatography
hTH Human Tyrosine Hydroxylase
kDa kilodalton
KORP Knowledge-based ORientational Potential
kV kilovolts
LC Liquid chromatography
L-DOPA 3,4-dihydroxyphenylalanine
MAPKAPK2 MAP kinase-activated protein kinase 2
MRE Molar residue ellipticity
MS Mass spectrometry
N-terminal Amino terminal
NA Noradrenaline
NMR Nuclear Magnetic Resonance
NS Negative staining
OD Oligomerization domain
PAH Phenylalanine Hydroxylase
PDPK 3-phosphoinositide-dependent protein kinase
PKA Protein kinase A
PP2A/C Protein phosphatase 2A/C
PRAK p38-regulated/activated protein kinase
RD Regulatory domain
SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
SN Substantia nigra
SPA Single particle analysis
TEM Transmission electron microscopy
TEV Tobacco etch virus
TH Tyrosine Hydroxylase
THD Tyrosine Hydroxylase Deficiency
TPH Tryptophan Hydroxylase
v/v volume/volume
w/v weight/volume
XL Crosslink
L IST OF A MINO A CIDS AND L ETTER C ODE
Alanine Ala, A Leucine Leu, L
Arginine Arg, R Lysine Lys, K
Aspartic acid Asp, D Methionine Met, M Asparagine Asn, N Phenylalanine Phe, F
Cysteine Cys, C Proline Pro, P
Glutamic acid Glu, E Serine Ser, S
Glycine Gly, G Tyrosine Tyr, Y
Glutamine Gln, Q Threonine Thr, T
Histidine His, H Tryptophan Trp, W
Isoleucine Ile, I Valine Val, V
I NTRODUCTION
1. I
NTRODUCTION1.1. AROMATIC AMINO ACID HYDROXYLASES
Phenylalanine hydroxylase (PAH), tyrosine hydroxylase (TH) and tryptophan hydroxylases 1 and 2 (TPH1, 2) comprise a family of pterin-dependent enzymes termed aromatic amino acid hydroxylases (AAAHs). They are non-heme iron-dependent monooxygenases that catalyze the hydroxylation of the aromatic amino acids L-phenylalanine (Phe), L-tyrosine (Tyr) and L-tryptophan (Trp) using (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) as cofactor and dioxygen as additional co-substrate (Fitzpatrick, 1999; Skjaerven et al., 2014).
While PAH is mainly involved in the catabolic metabolism of Phe through its hydroxylation to Tyr, the other AAAHs participate in anabolic pathways by making precursors for catecholamines (CAs) (TH) as well as serotonin and melatonin (TPHs).
Tyr is hydroxylated by TH to produce 3,4-dihydroxyphenylalanine (L-DOPA; DOPA from now on), whereas the TPHs hydroxylate Trp to 5-OH-Trp (Figure 1A) (Skjaerven et al., 2014).
CAs are a group of monoamines that includes dopamine (DA), noradrenaline (NA) and adrenaline (AD). DA is the precursor of NA and AD and contains a catechol core (Figure 1B). The CAs were first described as hormones and later discovered to act also as neurotransmitters (Nagatsu & Nagatsu, 2016). Since the final products of TH activity have this dual function, it is essential to keep a good homeostatic balance and regulation of the metabolism to avoid any disturbance of the normal organism functionality. Thus, the biosynthesis of CAs and its regulation is crucial, as they play a role in many different and versatile pathways, notably as neurotransmitters in the brain. Some of the processes where CA play important roles are motor control, emotions, reward, biorhythms, learning, blood pressure and lactation. Therefore, TH has to be regulated in many different cellular environments to fulfill the wide variety of physiological functions linked to neurotransmission or hormone signaling (Klein et al., 2019).
Figure 1. The function of aromatic amino acid hydroxylases (AAAHs). (A) Schematic reactions catalyzed by the different AAAHs. They all carry out the hydroxylation of an aromatic amino acid in presence of iron, a reductant pterin cofactor (BH4) and molecular oxygen as co- substrate. (B) DA, NA and AD biosynthesis pathway.
Phenylalanine Hydroxylase (PAH)
Tyrosine Hydroxylase (TH)
Tryptophan Hydroxylase (TPH)
BH4 4α-OH-BH4
O2
BH4 4α-OH-BH4
O2
BH4 4α-OH-BH4
O2
Dopamine
Serotonin
BH4 = Tetrahydrobiopterin 4α-OH-BH4 = 4α-hydroxypterin
Phe Tyr
Tyr
DOPA
Trp 5-OH-Trp
A
B Dopamine
Noradrenaline Adrenaline
DBH
PNMT
AAAD= Aromatic Amino Acid Decarboxylase
DBH= Dopamine β-Hydroxylase PNMT= Phenylethanolamine N-Methyltransferase AAAD
DOPA
1.1.1. EVOLUTION OF THE AAAHS
The eukaryotic AAAHs family is suggested to arise from a common hydroxylase, with the catalytic domain of PAH and its function being the most probable ancestor.
This is indeed the only AAAH gene found in bacteria and gene duplication and intron losses and gains have contributed to the increase in complexity of these enzymes in eukaryotic organisms (Siltberg-Liberles et al., 2008). AAAHs are proteins consisting of several domains with different evolutionary characteristics, but their occurrence in distant phylogenetic groups emphasizes their relevance from a functional point of view (Cao et al., 2010). All the AAAHs have three domains: the N-terminal regulatory domain (RD), which contains a disordered region of different length at its N-terminus followed by a structural domain known as ACT domain (Aspartate kinase-Chorismate mutase-TyrA domain); the central catalytic domain (CD) which contains the active site;
and the oligomerization domain (OD) at the C-terminus, responsible for dimerization and tetramerization (Figure 2A). The CD is the largest and most conserved of the three domains, showing around 50-75 % of sequence identity and almost identical structure among the different enzymes. However, accumulative and critical mutations in this domain are responsible for evolution of the substrate specificity of each AAAH.
Whereas TH is able to catalyze the hydroxylation of the three aromatic amino acids, PAH and the TPHs barely have affinity for Tyr as substrate. Therefore, TH might have diverged from the ancestral enzyme and evolved to acquire this preference for Tyr hydroxylation (Daubner et al., 2000). On the other hand, the RD distribution in AAAH genes has been more dynamic since it is not present in prokaryotic genes, being gained later in evolution. This variability has contributed to generate a major functional divergence among the enzymes, such is the case with some eukaryotic species that do not have the RD domain, or the human TH, the only species showing four different isoforms produced by alternative splicing. So, evolution has generated different mechanisms to modulate the different hydroxylase activities (Cao et al., 2010;
Daubner et al., 2011; Pribat et al., 2010).
1.1.2. FUNCTION OF THE AAAHS
The three enzymes of this family are responsible for catalyzing the hydroxylation of an aromatic amino acid by a common reaction mechanism that will be described
below. By catalyzing the degradation of Phe to Tyr, which is the first reaction in the catabolic degradation of Phe, PAH is responsible for removal of most of the Phe from the diet and protein catabolism, avoiding the neurotoxic accumulation of this amino acid. Phe degradation takes place in the liver, showing an activation by Phe, which binds with positive cooperativity to PAH (Flydal & Martinez, 2013; Skjaerven et al., 2014). However, TH and TPHs are the rate-limiting enzymes in the biosynthesis of CAs and serotonin neurotransmitters, respectively. Those are anabolic processes that take place in the central nervous system (CNS), but also in the peripheral nervous system, and in the particular case of TH in the adrenal medulla (Fitzpatrick, 1999). TPH1 and TPH2 are encoded by distinct genes, and while TPH1 is expressed in intestinal cells and the pineal body, TPH2 is mostly expressed in brain (Walther et al., 2003). Its product is 5-OH-Trp, the precursor of serotonin, a neurotransmitter important in several functions as CNS control, mood or sleep. Failure of the TPH regulation or mutations affecting the serotonergic system can lead to different mental disorders such as depression, drug abuse or anxiety (Teigen et al., 2007).
1.1.3. STRUCTURE OF THE AAAHS
Structurally, the AAAHs present a common homotetrameric arrangement and, as mentioned before, they all share the same three-domain organization. The CD is very conserved whereas the RD, and in particular its disordered N-terminal region, is responsible for the main differences in the family with regard to sequence, length and the regulatory mechanisms involved in modulating their activity (Figure 2A). PAH has been the most studied enzyme in this family, and recently the full-length structures of rat and human PAH were resolved by X-ray crystallography (Arturo et al., 2016; Flydal et al., 2019; Meisburger et al., 2016). The structure of the human enzyme was also obtained by cryo-electron microscopy (cryo-EM) (Flydal et al., 2019). These structures confirm the arrangement of the RDs in the resting tetrameric PAH structure with and without BH4 (Figure 2B). On the other hand, the atomic structures of the full-length TH and TPH (Figure 2C) have not been determined and the only available structures are those of the CD and OD, and of the RD in different structures. Low resolution structures of full-length TH places the RDs as dimers (Bezem et al., 2016) or separated (Szigetvari et al., 2019).
Figure 2. Overall domain structure of the AAAHs. (A) Schematic domain arrangement of the three different AAAHs. (B) Atomic structure of the PAH showing the non-dimeric arrangement of the RD (dark blue, pointed with red arrows) in regard to the CD (light blue) and OD (cyan) (PDB 6HYC). (C) The CD (light green) and OD (dark green) structure of the TPH2 (PDB 4V06).
The RD has not been solved by any structural technique. (D) A view of the TH active site showing the conserved arrangement of the iron-binding “2-His-1-carboxylate facial triad”.
(E) The flexible loop and other residues involved in substrate recognition and binding are highlighted in yellow. The Asp425 (in green) is an important residue in TH, maintaining the environment for substrate binding. (F) Residues involved in the proper positioning of the cofactor BH4 in the active site. BH4 is essential in the hydroxylation reaction. All the specific residues in panels (D), (E) and (F) follow the numeration of the TH sequence (PDB 1TOH). The location and residues in PAH and TPHs are conserved.
A
F D
10 Å
Asp189
Asp328
Asp425
Cys177 Arg316
E
FeII H2O 5 Å His331
Glu376
Tyr371 Phe300
BH4 Glu332 Leu295
Glu376 His331
His336
3 Å
B
C
His336 Regulatoy
domain (RD)
Catalytic domain
(CD)
Oligomerization domain
(OD)
Tail ACT
The N-terminal size varies resulting in different RDs lengths from 100 to 160 residues with a low identity among the different AAAHs (<14%) and increased complexity. As mentioned before, human TH, as a consequence of alternative splicing, results in four different isoforms with different length. The human TH isoform 1 (TH1) would correspond to the rat version of the enzyme (93% similarity). The differences in the N-terminal region and the variability in TH are the cause of the wide regulatory mechanisms exerted to control their function. The CD has approximately 300 residues and contains the active site and is followed by the OD, composed of two b-strands and the a-helices that support the tetrameric structure of these enzymes (Figure 2A). The TH is a highly stable tetramer formed by a dimer of dimers, and this is also the case of the TPHs, whereas PAH is found in an equilibrium between dimer and tetramer. In the case of the isolated RD, it has also been observed that it exists in an equilibrium between the monomeric and dimeric structure and that the dimerization is driven by Phe for PAH, but is not dependent on the substrate in the case of TH (Fitzpatrick, 1999;
Goodwill et al., 1997; Kleppe et al., 1999; Patel et al., 2016; Roberts & Fitzpatrick, 2013; Zhang et al., 2014).
The most conserved residues in the AAAH family are those involved in the catalysis, and therefore, those found in the CD. The active site is surrounded by four helices rich in hydrophobic residues that create a proper environment for the aromatic amino acid substrate interaction. Four residues, a Pro, a His (that also interacts with the iron) a Phe and another Phe (or Trp), form this hydrophobic pocket. The iron, that must be in its ferrous state for the enzymes to be active, is located in this hydrophobic gap, coordinated in all cases with two His and a Glu residues, in different positions depending of the enzyme (Figure 2D). The reduction step from the ferric to the ferrous form is a process facilitated by the reducing activity of BH4. The pterin binding site, very conserved in the AAAHs, mainly interacts with the side chains of a Glu and a Phe, among others residues (Figure 2E). In the case of the amino acid substrate, this interacts mainly with an Arg and an Asp (Figure 2F) (Roberts & Fitzpatrick, 2013).
1.1.4. AAAHS SUBSTRATE SPECIFICITY
Despite the similarity of the active site pockets, some residues are determinant for the enzyme substrate specificity. TH is the only enzyme that can process Tyr to DOPA,
but it can also hydroxylate Phe or Trp, albeit at a slower rate. The TPHs hydroxylate Trp but could also hydroxylate Phe with similar enzyme kinetics values. In the case of PAH, apart from Phe it can also hydroxylate Trp, but neither TPH nor PAH have the ability to use Tyr as substrate. Some studies with rat hydroxylases have shown how point mutations in residues that are conserved for a specific AAAH, but are different in the other AAAHs, affect the affinity and specificity for the substrate. The residue Asp425 in TH (Ile366 in TPH or Val379 in PAH) is an important residue that, although not involved in the direct interaction with either the substrate or the cofactor, plays a crucial role in maintaining the structure of the active site and therefore, in the affinity for the specific substrate (Figure 2F). In TH, the D425V mutation results in the complete loss of the ability to hydroxylate Tyr to DOPA, while it increases the affinity for Phe (Fitzpatrick, 2003). Other mutations in TH boosting the affinity for Phe are H323Y or Q310H, albeit at a smaller rate (Daubner et al., 2006). On the other hand, the opposite mutations on PAH do not enhance the affinity for Tyr, but instead cause a decreasing affinity for Phe.
The only case in which an increased affinity for Tyr has been measured is for the double mutation H264Q and V379D in PAH, but never as high as the natural affinity for Phe (Fitzpatrick, 2003). Most of these important residues in the specific recognition of the substrate are found in a flexible loop located on top of the amino acid binding site (residues 423-428 in TH; 378-381 in PAH). Residues Arg316 and Asp328 in TH are also important in Tyr binding (Figure 2F), while Glu332 and Phe300 interact with BH4
(Figure 2E) (Arg270, Asp282, Glu286 and Phe254 in PAH, respectively) (Daubner et al., 2000). In the case of TPH, Phe313 (Tyr313 in PAH and TH) is important for Trp binding affinity. The mutation Phe to Trp increases the preference for Phe but not for Tyr (Skjaerven et al., 2014).
1.1.5. REGULATION OF AAAHS ACTIVITY
Regulatory and activation mechanisms are also different in each hydroxylase, mostly due to the divergent structures of their N-terminal RDs. TH modulation is by far the most complex one, considering the diverse isoforms that are found in human compared with other species and with the other two enzymes. TH regulation by phosphorylation or CA feedback inhibition will be further discussed. TPH regulation is not yet totally understood, and PAH regulation is based on its own substrate (Phe),
inhibition by BH4 and phosphorylation of Ser16 (Fitzpatrick, 2012). The regulation of PAH activity is crucial because since Phe is an essential amino acid, a certain amount must be preserved but in high amounts it can be neurotoxic. In the case of activity regulation mediated by BH4, it has been described how its absence induces loss of the TH activity, while it does not impair TPH function in the brain. In the case of PAH, this cofactor acts by stabilizing and inhibiting the enzyme function (Thöny et al., 2008). The N-terminal region in PAH is an autoregulatory region (IARS) that can be phosphorylated at Ser16 by cAMP-dependent protein kinase (PKA). The N-terminal disordered region undergoes conformational changes that may participate in the activation/deactivation of the enzyme. Whether TPH phosphorylation has any effect is still under study (Jaffe et al., 2013; Kobe et al., 1999; Winge et al., 2008). Mutations in the TPH gene are linked to psychiatric diseases like bipolar affective disorder or depression, while mutations in the PAH gene that lead to misfunction results in accumulation of Phe (hyperphenylalaninemia) in plasma and neurological damage, two characteristic failures in untreated phenylketonuria (Flydal & Martinez, 2013; Haavik et al., 2008).
1.1.6. CATALYTIC REACTION
The hydroxylation reaction is one of the most common reactions in the oxidative metabolism, catalyzing the conversion of a carbon-hydrogen into a carbon-hydroxyl bond. Biologically, this reaction is essential not only in the context of the aromatic amino acids metabolism, but in other pathways such as detoxification, drug activation/deactivation, and post-translational modifications of proteins. All these different processes make this type of reaction a widespread one, used in industrial production and pharmacological processes (Holland & Weber, 2000).
Due to the high-level of homology in the CD of the AAAHs, the mechanism for amino acid hydroxylation is a common process. The reaction catalyzed by the four AAAHs takes place in two steps. The first is the rate-limiting step, which is the generation of a reactive hydroxylating intermediate, followed by the oxygen transfer to the amino acid.
The inactive enzyme is bound to FeIII, so before the amino acid substrate binds to the enzyme, BH4 interacts with the active site and carries out the reduction of ferric to
ferrous iron, resulting in the formation of an iron-pterin intermediate, which may be FeII-peroxypterin. BH4 binding results in the conformational change of the flexible loop located in the active site. This structural change provides the arrangement of the hydrophobic pocket for the substrate binding. Before BH4 reacts with the oxygen and the iron, the amino acid substrate has to be bound to the active site.
Figure 3. Schematic reaction mechanism of the AAAHs. On the left, the non-heme iron at the active site shows the known coordination called “2-His-1-carboxylate facial triad”, which in TH1 correspond to His331, His336 and Glu376, and three water molecules. When FeIII is bound, the enzyme is not able to carry out the reaction, so binding of BH4 is necessary to reduce it to FeII. In the center, BH4 reduces the iron and then forms the FeII-peroxypterin intermediate only in presence of the substrate. Cleavage of the O-O bond by one of the molecular waters in the active site results in the release of the hydroxylated BH4 and formation of the hydroxylating intermediate. On the right, the reactive FeIVO promotes the insertion of molecular oxygen into the substrate leading to the release of the hydroxylated product.
The presence of the molecular oxygen as a co-substrate would induce the formation of a bond between the BH4 and the iron resulting in the FeII–peroxypterin intermediate. A heterolysis of the O-O bond by a water molecule in this intermediate, favors the formation and parallel release of a reactive enzyme-FeIVO intermediate and 4a-hydroxypterin (Bassan et al., 2003). The FeIVO hydroxylating intermediate is the species that some studies have detected in this type of reactions, although previous intermediates to the FeIVO form are still under study (De Visser, 2010; Eser et al., 2007). This ferryl species mediates the amino acid hydroxylation by an electrophilic
His336
His331
Glu376 BH4
O2 substrate
OO BH4
4α-hydroxypterin
O hydroxylated product
FeII FeIV
FeIII H2O
aromatic substitution mechanism. The FeIVO promotes the insertion of one atom of molecular oxygen. This process implies the loss of aromaticity of the ring and then, the subsequent rearomatization after losing one hydrogen. After the catalysis reaction takes place, the hydroxylated pterin cofactor is released to be recycled by a dihydropteridine reductase to start a new hydroxylating cycle (Figure 3) (Fitzpatrick, 2003; Grüschow et al., 2019; Roberts & Fitzpatrick, 2013).
1.2. TYROSINE HYDROXYLASE
TH was discovered in 1964 as the enzyme that catalyzes the hydroxylation of Tyr to DOPA. This is the rate-limiting reaction in the synthesis pathway of CAs (Nagatsu et al., 1964) where DOPA is decarboxylated (by aromatic amino acid decarboxylase) to generate DA, which can be hydroxylated by dopamine-b-hydroxylase (DBH) to form NA, which can be subsequently methylated by phenylethanolamine-N- methyltransferase (PNMT) to form AD (Figure 1B). TH is considered a cytoplasmic enzyme, but it has been also found associated to the membranes and in the nucleus (Nakashima et al., 2016). Human TH (hTH) is the only AAAH existing as four isozymes (hTH 1 – 4) due to an alternative splicing mechanism from a single TH gene. In case of the primates, two isoforms are found, homologous to hTH1 and hTH2. However, in the rest of mammals a single form of TH is expressed (Haycock, 2002). The expression levels of each isoform are different depending on the location in the nervous system, which may be a symbol of the intricated regulation of TH and the subsequent CAs function and activity (Grima et al., 1987).
The differences between the splice variants are found in the RD, in particular in its disordered N-terminus, and have to do with insertions of different length between residues Met30 and Ser31 in hTH1. This variable length between TH forms does not affect its catalytic activity, but appears to produce significant differences in regulation.
The hTH1 isoform, the one without insertion, is the most similar to rat or mouse TH, while hTH2-4 have an insertion, after Met30 in hTH1, of 4, 27 and 31 residues, respectively (Figure 4) (Nagatsu & Nagatsu, 2016). The main location of hTH is the CNS, the peripheral sympathetic neurons and the adrenal medulla (Skjaerven et al., 2014), with hTH1 and hTH2 being the predominant in brain and the adrenal medulla (Cianchetta et al., 2010; Haycock, 1991; Lewis et al., 1993).
Figure 4. Human TH isoforms. Schematic representation of the four different splicing variants showing the main phosphorylation sites, and the insertions between the Met30 and Ser31 residues (adapted from Daubner et al., 2011).
Conversion of Tyr to DOPA and regulation of TH is essential in keeping the CA metabolism functional. Any alteration in TH gene expression will affect DA, NA and AD production, and thus distinct functions related with their activities as neurotransmitters and hormones. Mutations in the TH gene and protein deficiency are associated with genetic disorders such as the autosomal recessive form of “L-dopa- responsive dystonia” (DRD) or “infantile parkinsonism”, which are now collectively known as TH deficiency (Willemsen et al., 2010). Furthermore, a decrease of TH activity and protein is an early biochemical feature in Parkinson’s disease (PD), caused by the death of dopaminergic neurons.
1.2.1.STRUCTURE OF TH
TH is a homotetrameric enzyme formed by a dimer of dimers with an approximate molecular weight of 240 kDa. The human TH isoform 1 (hTH1) contains the RD (residues 1-165) that includes a disordered region (residues 1-70), the CD (166-456) and the OD (457-498) (Figure 5A). To date and due to the high flexibility of both the RD and its link with CD, only the atomic structures of truncated forms of the protein are available, such as the crystal structure of the CD and OD domains (PDB 1TOH)
S31 S40 S19
S19 S19 S19
VRGQ
GAPGPSLTGSPWPGTAAPAASYTPTPR
hTH1 hTH2 hTH3 hTH4
(Goodwill et al., 1997) or the dimeric structure of the isolated RD from rat, which was solved by NMR (PDB 2MDA) (Figure 5B) (Zhang et al., 2014).
The OD (40 residues) presents two b-strands and a long a-helix structure with a conserved hydrophobic core. This core consists of a heptad-repeat leucine rich region, which allows to stablish the tetramerization interface. The arrangement in this domain is defined by an antiparallel coiled coil assembly driving to form dimers through salt bridges and hydrogen bonds, and then, the tetramer is stabilized by leucine zippers.
The b-strands and the loop at the beginning of the OD might be adapting the subsequent helices position (Fitzpatrick, 1999; Flatmark & Stevens, 1999).
Figure 5. Structural organization of TH. (A) Schematic arrangement showing the different domains from the N-terminus to the C-terminus. The RD (pink) in human TH containing an unstructured tail at the first 70 residues and an ACT domain. The CD (dark magenta), performing the TH enzymatic activity and the C-terminal part form the OD for oligomerization (gray). (B) Truncated atomic structures of the human tetramer of TH comprising the CD (dark magenta) and the OD (gray) (PDB 2XSN). One of the subunits forming the tetramer is highlighted in light magenta and blue. On the right side, the atomic structure of the rat RD, solved by NMR (PDB 2MDA).
1 RD 166 CD 457 498OD
CD OD
CD 20 Å
RD
A
20 Å
90ºB
Tail ACTThe CD harbors the active site where the iron, the BH4 and the Tyr bind to catalyze the hydroxylation reaction in the presence of molecular oxygen. The CD is mainly a- helical (~ 49%), with the rest consisting of ~ 9% b-strand and ~ 42% long loops. There are two loops (residues 290-296 and 423-428) and four a-helices (297-304, 329-340, 343-356 and 361-372) surrounding the catalytic pocket (17 Å deep / 15 Å wide). The three amino acid coordinating the iron are His331, His336 and Glu376 (Figure 2D) (Goodwill et al., 1997, 1998). Those residues are highly conserved, and the region comprising residues 334 to 339 is aligned without any gap in all the eukaryotic enzymes. This kind of coordination with the metal atom is known as “2-His-1- carboxylate facial triad”. The iron is situated at the bottom of the active site cleft (10 Å deep) and in absence of substrate is connected to three water molecules. The BH4
emplacement is established by interaction with residues Leu294, Leu295, Phe300, Tyr371, Glu376 and with one of the iron ligated water (Figure 2E) (Teigen et al., 2007).
The molecular oxygen position that catalyzes the reaction would be located between the iron and the pterin cofactor. It has been shown how residues comprising the loop 179-189 play a main role in the amino acid substrate positioning (Daubner et al., 2006). Mutations involving residues in this loop result in modifications of the kinetics values for the amino acid substrate. The decrease in Tyr hydroxylation level shows the importance of the side-chains of residues in this loop in creating the proper environment for the substrate binding. Furthermore, this loop is quite flexible, so its movement to interact with the amino acid substrate will place it in the optimal location for its reaction with the ferryl hydroxylating intermediate. Asp425 was identified as an important amino acid in substrate specificity, but also Arg316 and Asp328 were shown to be involved in Tyr binding (Figure 2F) (Daubner et al., 2006;
Fitzpatrick, 2003; Skjaerven et al., 2014).
Most of the regulation mechanisms controlling TH activity are located at the N- terminal amino acid sequence, the most variable region having different length not only among human isoforms but also with other species. Although it is clear that the main role of this domain is to control TH activity, and hence all the catecholaminergic pathways, experiments deleting the first 165 residues have shown that this domain is not essential to maintain the enzyme activity, and the same can be said for residues connecting the RD and CD (165-175) (Kumer & Vrana, 1996).
The RD has four specific phosphorylation sites located in the N-terminal tail in positions Ser8 (Thr8 in humans), Ser19, Ser31 and Ser40 (Dunkley et al., 2004). The first 70 amino acids make a flexible unstructured tail which has been predicted to contain an a-helix of about 20 residues around residues 37-58 (Buchan & Jones, 2019;
Yang & Zhang, 2015). Part of this region, the one encompassing residues 40-49, is very conserved across different TH species, which probably has a functional evolutionary significance (Zhang et al., 2014). Some studies point to phosphorylation at Ser40 as a crucial modification that could affect the assembly of the first 70 residues, promoting the accessibility or closure of TH active site (Wang et al., 2011). The flexible N-terminus is followed by the characteristic regulatory ACT domain formed by four b-strands and two a-helices. Since TH is not known to be an allosteric enzyme, and the ACT domain is typical of many allosteric enzymes, it is suggested that TH has lost the intrinsic ACT function, maintaining the structure throughout the evolution (Grant, 2006; Zhang et al., 2014).
1.2.2.FUNCTION AND REGULATION
As it has already been mentioned, the main TH function is to catalyze the rate- limiting step in DA synthesis. Regulation mechanisms to maintain its intracellular stability are quite sophisticated due to its pivotal role in the CAs biosynthesis. The specificity in the modulation mechanisms at the N-terminal unstructured tail provides a broad framework for both regulation and function of TH activity. The processes that modulate the activity not only involve the N-terminal region and can be divided in two main categories: a short-term regulation including allosteric regulation, feedback- inhibition and phosphorylation; and a medium-long term modulation of the gene expression.
Allosteric regulation is performed by heparin and polyanions or phospholipids, in such a way that these effectors decrease the Km for BH4, increasing TH activity. But all those interactions have only been demonstrated in vitro, and it is not clear whether it has an important modulating effect in vivo (Kumer & Vrana, 1996).
Short-term modulation by feedback-inhibition is carried out by the end-products of the pathway, CAs, which ensure that TH will not suffer an irreversible inactivation.
When TH is bound to FeIII, it can bind either BH4 or DA (Ramsey & Fitzpatrick, 2000). DA
binding produces the enzyme activity inactivation by competing with BH4, and also it has been shown how it changes the iron from the ferrous to the ferric form. This binding is a reversible process that depends on their intracellular concentrations (Urano et al., 2006). Binding of DA involves an interaction with the regulatory domain, in such a way that thermal and kinetic stabilities of the enzyme are also increased (Daubner et al., 2011). In previous studies it has been shown how TH is less susceptible to cleavage by trypsin in the presence of DA, confirming that CA binding stabilizes the enzyme (McCulloch & Fitzpatrick, 1999). However, an excess of CA could also promote the release of iron and consequently TH destabilization (Martínez et al., 1996).
BH4 acts not only as a cofactor in the catalysis of the hydroxylation reaction but also indirectly regulates the CAs biosynthesis. This occurs not only by competing with DA in the binding to the TH active site, but also by having a chaperone-like function.
This cofactor would increase TH synthesis and also its binding to late-stage protein folding intermediates could improve the folding process or revert misfolding of the protein. For that reason, BH4 has been used as supplementation to stimulate TH activity in dopaminergic neurons and to increase the enzyme stabilization (Thöny et al., 2008). On the other hand, an excess of BH4 could also result in TH inhibition (Alterio et al., 1998) whereas low intracellular concentrations of the cofactor would induce a decrease of CAs production and consequently TH destabilization and possible aggregation (Urano et al., 2006).
The most important regulatory mechanism, together with the feedback inhibition by CAs, is based on the phosphorylation/dephosphorylation of the N-terminal tail at residues Ser/Thr8, Ser19, Ser31 and Ser40. Each residue is phosphorylated in vivo by a specific kinase and is involved in regulating a different cellular process.
Dephosphorylation, on the other hand, is not so specific and is carried out by two serine phosphatases, PP2A and PP2C (Protein phosphatase 2A/C) (Dunkley et al., 2004). In Ser/Thr8, the kinases involved in its phosphorylation are PDPK (a3- phosphoinositide-dependent protein kinase), ERK1 (extracellular signal-regulated kinases) and ERK2, but its phosphorylation has not been associated to TH activity regulation. Ser19 is phosphorylated mainly by CaMPKII (Ca2+ /calmodulin-dependent protein kinase II) and also by PRAK (p38-regulated/activated protein kinase), and it is linked to an increase of calcium concentration in the cell. The MAP (mitogen-activated
protein) kinases ERK1/2 and Cdk5 (Cell division protein kinase 5) have been shown to act upon Ser31 (Salvatore, 2014); while in Ser40, the most studied together with Ser19, the kinases implicated are mainly PKA (Protein kinase A) and MAPKAPK2 (MAP kinase-activated protein kinase 2) (Figure 6) (Daubner et al., 2011; Dunkley et al., 2004; Dunkley & Dickson, 2019).
Figure 6. Regulation of TH by phosphorylation. Specific kinases that phosphorylate the different residues at the TH N-terminal, and their consequent function.
The alternative roles in TH activation of each different phosphorylated residue are still under study. It has been shown in different tissues how a hierarchical strategy exists in the phosphorylation process. An increase of the intracellular calcium concentration acts as a signal to activate Ser19 phosphorylation by CaMPKII (Ca2+/calmodulin-dependent protein kinase II), and this action precedes Ser40 phosphorylation by the same kinase, in such a way that TH activity increases.
Otherwise, prior phosphorylation of Ser40 does not increase or decrease Ser19 phosphorylation. Regularly, Ser31 phosphorylation is always slower than that of Ser19, but different stimulus or signal transduction pathways could give a preference to Ser31 phosphorylation. Then, phosphorylation at Ser40 could be controlled by either pSer19 or pSer31 (Dunkley et al., 2004; Dunkley & Dickson, 2019; Jorge-Finnigan et al., 2017).
Phosphorylation at Ser19 (pSer19TH) is related with different actions or locations of TH. pSer19TH has been shown to induce structural changes in the RD (Bevilaqua et al., 2001). Crystallographic and molecular dynamics simulations have shown how it is critical in the high affinity interaction of a TH peptide (N-terminal residues 1-43) with the regulatory 14-3-3 adaptor protein. This interaction is still under study, but it seems to participate in stabilizing TH by avoiding kinases/phosphatases action or relocating TH inside the cell (Kleppe et al., 2014; Skjevik et al., 2014). Another mechanism to maintain the intracellular TH homeostasis is through degradation. Some studies have associated pSer19TH with the targeting for proteasome-mediated degradation in the nucleus. However, it is still unknown whether TH phosphorylation takes place before or after nucleus import from the cytoplasm (Nakashima et al., 2016, 2011).
In the case of Ser31 phosphorylation (pSer31TH), it has been recently demonstrated its role in TH transport from the cell soma to the terminals through the microtubules. pSer31TH has been shown to impair Ser40 phosphorylation, in a way that more CAs can be bound, inactivating and stabilizing TH during its transport (Jorge- Finnigan et al., 2017). pSer31TH is also involved during TH protein loss in PD, where some compensatory mechanisms take place in order to restore TH activity in the dopaminergic neurons (Shehadeh et al., 2019). It has been demonstrated how just pSer31TH has an influence in DA recovery in the substantia nigra (SN), but not in the striatum. Therefore, this differential regulation could be linked to extracellular-signal- regulated kinase (ERK) activation, and neither Ser40 nor Ser19 phosphorylation have a correlation with DA recovery. Furthermore, pSer19TH or pSer40TH have been associated to TH loss in striatum by proteasome-mediated degradation (Nakashima et al., 2011; Salvatore, 2014).
TH inhibition by DA binding is tightly linked to the subsequent activation by phosphorylation at Ser40. This process results in decreasing the Km value of BH4 and also the affinity for CAs. Furthermore, pSer40TH has been associated to the possibility of promoting the open conformation of the enzyme through the conformational change in the N-terminal tail. The open form would allow the rearrangement of the N- terminus to give access to the active site leading the release of the DA. This process also takes place in a calcium-dependent manner (Bezem et al., 2016; Daubner et al.,
2011; Martínez et al., 1996; Ramsey & Fitzpatrick, 2000; Salvatore et al., 2001; Toska et al., 2002; Wang et al., 2011).
Otherwise, full-length unphosphorylated TH has also been found interacting with membranes at nerve endings and synaptic vesicles. Studies using a truncated TH lacking part of the N-terminal domain have demonstrated the importance of this region in membrane binding. The significance of this interaction is not clear but it has been hypothesized that it could have a function in locating the DA production close to its packaging into the synaptic vesicles (Daubner et al., 2011; Kleppe et al., 2014;
Skjevik et al., 2014; Thórólfsson et al., 2002).
In the medium/long term regulation, there is a flexible mechanism that controls gene expression in different tissues attending to the different CA requirements.
Constant impulses in dopaminergic neurons activate TH gene transcription, increasing mRNA concentrations and TH protein in cells. Furthermore, modulation of alternative splicing is another way to regulate the CAs production in different physiological conditions such as alertness, stress responses or mood (Kumer & Vrana, 1996; Tank et al., 2008).
1.2.3. DYSFUNCTION AND ASSOCIATED DISEASES
There are some missense mutations in the TH gene and its promoter region that are associated to tyrosine hydroxylase deficiency (THD). Those disease-causing mutations are found in the CD. Mutations involving the RD do not lead to any disease.
THD is also known as “Segawa syndrome”, “infantile parkinsonism” or DRD (Table 1).
Differences between each one of these genetic diseases depends on the type of mutation and the phenotype expressed. “Segawa syndrome” for instance, also presents a defect related with GTP cyclohydrolase I mutations, which is involved in BH4
biosynthesis. THD is then a neurometabolic disorder characterized by a CA deficiency in the brain and, in many cases, patients can be treated with DOPA to revert the symptoms. Most of the mutations causing DRD affect the TH structure and therefore its function. They are usually located at the active site and impair the substrate or cofactor binding. L205P or G381L are examples of point mutations causing DOPA responsive parkinsonism or Segawa’s syndrome, respectively. Whereas in the first case, the mutation destabilizes the protein arrangement disrupting an a-helix
structure, in the second one the affinity for the amino acid substrate is decreased compared with the wild-type (Fossbakk et al., 2014; Goodwill et al., 1997; Ichinose et al., 1999; Korner et al., 2015; Willemsen et al., 2010).
Genetic dysfunction
Autosomal dominant hereditary progressive and Dopa- responsive dystonia (AD-HPD/DRD) (Segawa’s syndrome) Autosomal Recessive Dopa-Responsive Dystonia (AR-DRD):
Tyrosine hydroxylase deficiency (THD)
Autosomal recessive juvenile parkinsonism (AR-JP) Diseases associated
Parkinson’s disease Huntington’s disease Schizophrenia
Attention deficit / Hyperactivity disorder Addiction
Table 1. TH genetic dysfunctions and associated diseases.
Dysfunction or death of dopaminergic neurons are also associated with some diseases such as PD, Alzheimer disease or cardiovascular disease (Table 1) (Klein et al., 2019). While deficiency of DA in striatum is linked to movement disorders; non-motor symptoms such as depression or insomnia are caused by a lack of NA. Decreasing TH activity in DA neurons, or DA synthesis reduction may provoke the dopaminergic neurons death. Furthermore, DA is a very reactive compound by itself and is exposed to oxidative damage, driving also the death of dopaminergic neurons. In other cases, DA accumulation results in production of toxic compounds that cannot be well metabolized and lead to oxidative stress or to the formation of neurotoxic oligomers of a-synuclein (Daubner et al., 2011; Hare & Double, 2016). To compensate for the DA excess, TH can be phosphorylated at Ser19, and so targeted for proteasome-mediated degradation. However, in PD the proteasome pathway is also affected, increasing the symptomatology and severity of the disease (Nakashima et al., 2016).
1.3. THE RD, A KEY FACTOR IN TH MODULATION
In order to maintain an optimal biological activity and to enhance the organism survival, cell mechanisms have evolved to be able to respond to any environmental change. Maintaining cognitive and motor functions involves the coordinated action of each constituent in the cell that contributes to preserve neurological health. The different regulatory systems are part of this broad machinery intended to keep the cell balance. AAAHs are a small enzyme family that catalyzes the hydroxylation of amino acids that are essential in different metabolic pathways, from the degradation of neurotoxic Phe to the synthesis of important neurotransmitters in the CNS.
This work will be focused in achieving a deeper understanding of the TH structure and function. The structure of any protein is directly related with its biological function; thus, the elucidation of TH structure is important to unravel its mechanism and regulation. The structures of the different TH domains have been determined by X-ray crystallography and/or NMR separately (Goodwill et al., 1997; Zhang et al., 2014). However, the high flexibility of the RD N-terminal domain and the linker between RD and CD have been a handicap for biochemical and structural studies throughout the years. Regulation of TH activity is focused mainly on the phosphorylation sites found in the unstructured tail, but to date no structural technique has proven successful to determine the arrangement between the RD and CD.
O BJECTIVES
2. O
BJECTIVES1) To identify the best conditions for sample preparation that allow the generation of the highest-resolution structures by cryo-electron microscopy.
2) To determine the high-resolution structures of TH in the absence or presence of DA, and to describe in molecular terms the differences in the two structures.
M ATERIALS
AND M ETHODS
3. M
ATERIALS ANDM
ETHODS3.1. BIOLOGICAL MATERIAL AND REAGENTS
The samples analyzed in this work were provided by Dr. Aurora Martínez (Bergen University, Norway). Human TH1 (from now on referred as TH) and the different mutants were overexpressed in bacteria and purified on amylose resin as described for the His-MBP-TH in Bezem et al.,2016. Two mutants were generated lacking the first N- terminal 43 residues (THDN43) and 70 residues (THDN70). The fusion proteins expressed and used in this work contain a hexahistidine-tagged maltose-binding protein at the N-terminus (His6-MBP-TH) and a TEV protease cleavage site between His6-MBP and TH. The His6-MBP-TH proteins were first purified by affinity chromatography using amylose resin. The purified proteins were then treated with TEV protease to cleave off the N-terminal tag and a final size exclusion chromatography was performed to isolate the population containing the TH tetramers. The samples were kept in purification buffer (200 mM NaCl, 20mM Hepes pH=7.0) in all the experiments, unless stated otherwise. Buffers were degassed applying bubbling helium prior to sample preparation. The helium dissolves and displaces the dissolved air in the buffer, avoiding its presence and the oxidation of the different oxygen-sensitive compounds.
Commercial dopamine hydrochloride (DA) from Sigma-Aldrich (#H8502) was used to study the structure of TH in its presence. It was stored at 10 mM concentration and further diluted depending on the experimental requirements.
3.2. DOPAMINE BINDING
Once TH was purified and its functionality assessed, the protein was incubated with iron and DA to obtain the protein complex for further cryo-EM analysis. Ferrous ammonium sulphate (FAS) (Sigma-Aldrich #215406) was dissolved in degassed water. A final concentration of 8 µM FAS was added to TH and incubated for 2 min before DA addition (1.5:1 DA:TH stoichiometry) and subsequent incubation for 3 min before cryo- EM grid preparation and/or circular dichroism spectroscopy (CDS) assays.
3.3. SAMPLE PREPARATION FOR ELECTRON MICROSCOPY 3.3.1. NEGATIVE STAINING GRIDS PREPARATION
300 mesh grids (Maxtaform Cu/Rh HR26) coated with a thin (~ 6 nm) carbon layer were subjected to glow-discharge (15 s at 25 mA; EMITECH K100K), prior to the sample incubation, to increase carbon hydrophilicity. Aliquots of 5 µl of the different samples at the proper concentration were applied onto the grids and incubated for 1 min. After incubation, the excess of sample was removed by blotting with Whatman paper and the grids were stained for 1 min with 2 % (w/v) uranyl acetate and air-dried before TEM analysis. Images were recorded at 0° tilt in a JEOL JEM 1011 TEM, operated at 100 kV, using a Gatan ES1000W CCD camera at 100,000x nominal magnification.
3.3.2. CRYO-EM GRIDS PREPARATION
Purified TH (from now on referred as apo-TH when in absence of DA; and as TH(DA) when bound to DA) sample was subjected to a second size exclusion chromatography step to ensure protein homogeneity and stability. The sample was loaded onto a Superdex 200 Increase 3.2/300 (GE Healthcare) equilibrated with the same buffer at 4 ºC. Fractions of 30 µl were collected in an ÄKTAmicro (GE Healthcare) device. Cryo-EM grids were immediately vitrified using a Vitrobot Mark IV (FEI). Quantifoil UltraAufoil 1.2/1.3 300 mesh grids were previously glow- discharged for 30 s at 15 mA. Aliquots of 3 µl of the different samples were incubated onto the grids, blotted for 1 s at 4 ºC and 95% humidity and plunged into liquid ethane.
The TH(DA) sample was directly vitrified in the same conditions with no extra size exclusion chromatography step.
3.4. CRYO-EM 3.4.1. DATA ACQUISITION
All the samples were first checked using a 200 kV FEI Talos Arctica equipped with a Falcon III direct electron detector at the Centro Nacional de Biotecnología (CNB) cryo-EM facility.